Expression and secretion of heterologous polypeptides from caulobacter

ABSTRACT

DNA constructs are provided which code for a chimeric protein in which the C-terminal region corresponds to the extreme C-terminal amino acids of a Caulobacter S-layer protein, fused with a heterologous polypeptide. Bacterial cells containing the DNA constructs, or which express the DNA constructs and secrete the resulting protein, are provided. Chimeric proteins including the C-terminal amino acids of a Caulobacter S-layer protein are provided, including proteins which include antigenic epitopes of the Infectious Hematopoietic Necrosis Virus.

This application is a national stage application of PCT/CA97/00167,filed Mar. 10, 1997, which is a continuation-in-part of U.S. Ser. No.08/614,377, filed Mar. 12, 1996, now U.S. Pat. No. 5,976,864, which is acontinuation-in-part of U.S. Ser. No. 08/194,290, filed Feb. 9, 1994,now U.S. Pat. No. 5,500,353, which is a continuation-in-part of U.S.Ser. No. 07/895,367, filed Jun. 9, 1992, now abandoned.

FIELD OF THE INVENTION

This invention relates to the expression and secretion of heterologouspeptides, from Caulobacter wherein the heterologous polypeptide is fusedwith the surface layer protein (S-layer protein) of the bacterium, or aportion of the S-layer protein.

BACKGROUND OF THE INVENTION

Bacterial surface proteins have been used as carriers for foreign(heterologous) polypeptides (particularly in Salmonella and E. coli) forvarious purposes, including the development of live vaccines. In someinstances, the heterologous material is expressed as a fusion productwith a surface protein of the bacterium. Generally, the use of suchsurface proteins as a vehicle for expression and/or presentation ofheterologous polypeptides has been limited by the characteristics of aparticular surface protein. The lipopolysaccharide layer of a bacterium,which tends to stimulate a strong immune response, covers the integralouter membrane proteins of the organism and potentially affectsefficient presentation of a cloned epitope. Where the surface protein isfunctional (for example, as part of a filamentous portion of a bacterialcell surface) there will be limited opportunities to express a fusionproduct and still retain the surface protein's function. Generally, theorganisms that have been used for these purposes have been chosenbecause of the advantages presented in respect of the organism'srelationship to its host.

Many genera of bacteria assemble layers composed of repetitive,regularly aligned, proteinaceous sub-units on the outer surface of thecell. These layers are essentially two-dimensional paracrystallinearrays, and being the outer molecular layer of the organism, directlyinterface with the environment. Such layers are commonly known asS-layers and are found on members of every taxonomic group of walledbacteria including: Archaebacteria; Chlamydia; Cyanobacteria;Acinetobacter; Bacillus; Acuaspirillum; Caulobacter; Clostridium;Chromatium. Typically, an S-layer will be composed of an intricate,geometric array of at least one major protein having a repetitiveregular structure. In many cases, such as in Caulobacter, the S-layerprotein is synthesized by the cell in large quantities and the S-layercompletely envelopes the cell and thus appears to be a protective layer.

Caulobacter are natural inhabitants of most soil and freshwaterenvironments and may persist in waste water treatment systems andeffluents. The bacteria alternate between a stalked cell that isattached to a surface, and an adhesive motile dispersal cell thatsearches to find a new surface upon which to stick and convert to astalked cell. The bacteria attach tenaciously to nearly all surfaces anddo so without producing the extracelluar enzymes or polysaccharide“slimes” that are characteristic of most other surface attachedbacteria. They have simple requirements for growth. The organism isubiquitous in the environment and has been isolated from oligotrophic tomesotrophic situations. Caulobacters are known for their ability totolerate low nutrient level stresses, for example, low phosphate levels.This nutrient can be limiting in many leachate waste streams, especiallythose with high levels of iron or calcium.

All of the freshwater Caulobacter that produce an S-layer are similarand have S-layers that are substantially the same. Such S-layers appearsimilar by electron microscopy with the layer being hexagonally arrangedin all cases with a similar centre—centre dimension (see: Walker, S. G.,et al. (1992). “Isolation and Comparison of the Paracrystalline SurfaceLayer Proteins of Freshwater Caulobacters” J. Bacteriol. 174:1783-1792). 16S rRNA sequence analysis of several S-layer producingCaulobacter strains suggest that they group closely (see: Stahl, D. A.et al (1992) “The Phylogeny of Marine and Freshwater CaulobactersReflects Their Habitat” J. Bacteriol. 174:2193-2198). DNA probing ofSouthern blots using the S-layer gene from C. crescentus CB15 identifiesa single band that is consistent with the presence of a cognate gene(see: MacRae, J. D. and, J. Smit. (1991) “Characterization ofCaulobacters Isolated from Wastewater Treatment Systems” Applied andEnvironmental Microbiology 57:751-758). Furthermore, antisera raisedagainst the S-layer protein of C. crescentus strain CB15 reacts withS-layer proteins from other Caulobacter (see: Walker, S. G. et al (1992)[supra]). All S-layer proteins isolated from Caulobacter may besubstantially purified using the same extraction method (pH extraction)which would not be expected to be a general purpose method for otherbacterial membrane or surface associated proteins. All strains appear tohave a polysaccharide reactive with antisera reactive against CB15lipopolysaccharide species which appears to be required for S-layerattachment (see: Walker, S. G. et al (1992) [supra]).

The S-layer elaborated by freshwater isolates of Caulobacter are visiblyindistinguishable from the S-layer produced by Caulobacter crescentusstrains CB2 and CB15.

The S-layer proteins from the latter strains have approximately 100,000m.w. although sizes of S-layer proteins from other species and strainswill vary. The protein has been characterized both structurally andchemically. It is composed of ring-like structures spaced at 22nmintervals arranged in a hexagonal manner on the outer membrane. TheS-layer is bound to the bacterial surface and may be removed by low pHtreatment or by treatment with a calcium chelator such as EDTA.

The similarity of S-layer proteins in different strains of Caulobacterpermits the use of a cloned S-layer protein gene of one Caulobacterstrain for retrieval of the corresponding gene in other Caulobacterstrains (see: Walker, S. G. et al (1992) [supra]; and, MacRae, J. D. etal (1991) [supra].

Expression, secretion and optionally, presentation, of a heterologouspolypeptide as a fusion product with the S-layer protein of Caulobacterprovides advantages not previously seen in systems using organisms suchas E. coli and Salmonella where fusion products of other kinds ofsurface proteins have been expressed. All known Caulobacter strains arebelieved to be harmless and are nearly ubiquitous in aquaticenvironments. In contrast, many Salmonella and E. coli strains arepathogens. Consequently, expression and secretion of a heterologouspolypeptide using Caulobacter as a vehicle will have the advantage thatthe expression system will be stable in a variety of outdoorenvironments and may not present problems associated with the use of apathogenic organism. Furthermore, Caulobacter are natural biofilmforming species and may be adapted for use in fixed biofilm bioreactors.The quantity of S-layer protein that is synthesized and is secreted byCaulobacter is high, reaching 12% of the cell protein. The uniquecharacteristics of the repetitive, two-dimensional S-layer would alsomake such bacteria ideal for use as an expression system, or as apresentation surface for heterologous polypeptides. This is desirable ina live vaccine to maximize presentation of the antigen or antigenicepitope. In addition, use of such a presentation surface to achievemaximal exposure of a desired polypeptide to the environment results insuch bacteria being particularly suited for use in bioreactors or ascarriers for the polypeptide in aqueous or terrestrial outdoorenvironments.

SUMMARY OF THE INVENTION

This invention pertains to the discovery that the C-terminal region ofthe Caulobacter S-layer protein is essential for secretion of theS-layer protein. The inventors have determined that the 3′ region of thegene which encodes the C-terminal region of the S-layer protein isconserved among different strains of Caulobacter.

This invention provides a method of expressing and presenting to theenvironment of a Caulobacter, a polypeptide that is heterologous to theS-layer protein of the Caulobacter, which comprises inserting a codingsequence for the heterologous polypeptide in-frame into a S-layerprotein gene of Caulobacter, or a portion of said S-layer protein gene,whereby the polypeptide is expressed and secreted by the Caulobacter asa chimeric protein comprising the heterologous protein and all or partof the S-layer protein.

This invention provides a DNA construct for the aforemention chimericprotein, and a bacterium comprising such a DNA construct, wherein theDNA construct encodes all or part of a S-layer protein, and one or morein-frame sequences encoding one or more heterologous proteins.

This invention provides a DNA construct comprising one or morerestriction sites for facilitating insertion of DNA into the constructand, DNA encoding at least the 82 C-terminal amino acids of CaulobacterS-layer protein. Preferably, the C-terminal amino acids are orcorrespond to amino acids 944 or 945-1026 of the RsaA protein of C.crescentus.

This invention provides a DNA construct comprising DNA encoding aheterologous polypeptide sequence not present in a Caulobacter S-layerprotein upstream from and in-frame with DNA encoding at least the 82C-terminal amino acids of Caulobacter S-layer protein. Preferably, theC-terminal amino acids are or correspond to amino acids 944 or 945-1026of the rsaA protein of C. crescentus.

This invention also provides a secreted protein obtained from the cellsurface or cell medium of a Caulobacter cell expressing theaforementioned DNA constructs wherein the secreted protein comprises theheterologous polypeptide and at least the 82 C-terminal amino acids of aCaulobacter S-layer protein. Preferably, the C-terminal amino acids areor correspond to amino acids 944 or 945-1026 of the RsaA protein of C.crescentus.

DESCRIPTION OF THE DRAWINGS

For better understanding of this invention, reference may be made to thepreferred embodiments and examples described below, and the accompanyingdrawings in which:

FIG. 1 is the sequence of a Carrier cassette which may be cloned intothe PstI/BamHI site of pUC9 to deliver a gene sequence of interest tosites within a Caulobacter crescentus S-layer protein (rsaA) gene (SEQID NO:1).

FIG. 2 is a restriction map of a plasmid based promoter-less version ofthe rsaA gene (pTZ18U:rsaAΔP) containing restriction sites and which maybe used to accept heterologous DNA of interest.

FIG. 3 is the nucleotide sequence of linker BamHI-7165K (SEQ ID NO:2;and SEQ ID NO:3) carried in plasmid pUC9B (pUC7165K), which may be usedfor mutagenesis at sites created in rsaA by a specific or non-specificendonuclease.

FIG. 4 is the nucleotide sequence a linker BamHI-6571K (SEQ ID NO:4; andSEQ ID NO:5) carried in plasmid pTZ19 (pTZ6571K) which may be used formutagenesis at sites created in rsaA by a specific or non-specificendonuclease.

FIG. 5 is a map of insertion events at TaqI sites in the rsaA geneidentified by amino acid number of the insertion site in the S-layerprotein and scored according to whether the S-layer is produced in themodified organism.

FIG. 6 (comprising FIGS. 6a, b, and c) shows the complete nucleotidesequence of the C. crescentus S-layer (rsaA) gene (SEQ ID NO:6) and thepredicted translational product in the single letter amino acid code.The −35 and −10 sites of the promoter region as well as the start oftranscription and the Shine-Dalgarno sequence are indicated. Partialamino acid sequences determined by Edman degradation of rsaA protein andof sequenced peptides obtained after cleavage with V8 protease areindicated by contiguous underlining. The putative transcriptionterminator palindrome is indicated with arrowed lines. The regionencoding the glycine-aspartate repeats is indicated by underlined aminoacid code letters. This region includes five aspartic acids that may beinvolved in the binding of calcium ions.

FIG. 7 is a bar graph showing the approximate location by amino acidblock of 54 permissive sites in the rsaA gene corresponding to TaqI,HinPI, AciI, and MspI sites described in Example 3.

FIG. 8 is a portion of an amino acid sequence (SEQ ID NO: 8) from P.aeruginosa PAK pilin in which the 12 amino acid pilus peptide epitopereferred to in Example 5 is identified by superscript numerals 1-12.

FIG. 9 is the nucleotide coding sequence and corresponding amino acidsequence (SEQ ID NO:9) in respect of the 184 amino acid sequencecorresponding to amino acids 270-453 of the IHNV surface glycoproteindescribed in Example 6.

FIG. 10 is the amino acid sequence of the synthetic cadmium bindingpeptide referred to in Example 4. The cadmium binding site is shown inthe figure.

FIG. 11 shows locations of some of the sites in rsaA in which single andmultiple copies of the pilus peptide described in Example 5 wasexpressed and secreted as part of a chimeric rsaA protein.

FIG. 12 shows a portion of pUC8 containing various C-terminal fragmentsof rsaA as described in Example 7.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The preferred organism for use in this invention is Caulobacter,particularly C. crescentus. While similarity of the S-layer gene andS-layer secretion systems permits the use of any S-layer proteinproducing Caulobacter in this invention, C. crescentus strains CB2 andCB15 and variants of those strains which contain homologs of the geneencoding the 1026 amino acid paracrystalline S-layer protein describedin: Gilchrist, A. et al. 1992. “Nucleotide Sequence Analysis Of The GeneEncoding the Caulobacter crescentus Paracrystalline Surface LayerProtein”. Can. J. Microbiol. 38:193-208, are referred to in the examplesdescribed below.

Caulobacter strains which either are incapable of forming an S-layer,including those which shed the S-layer protein upon secretion, may beused in this invention. Examples are the variants CB2A and CB15AKSacdescribed in Smit, J., and N. Agabian. 1984. “Cloning of the MajorProtein of the Caulobacter crescentus Periodic Surface Layer: Detectionand Other Characterization of the Cloned Peptide by Protein ExpressionAssays”. J. Bacteriol. 160:1137-1145.; and, Edwards, P., and J. Smit.1991. “A Transducing Bacteriophage for Caulobacter crescentus Uses theParacrystalline Surface Layer Protein as Receptor”. J. Bacteriol. 173,5568-5572. Examples of shedding strains are CB15Ca5 and CB15Ca10described in Edwards and Smit (1991) [supra], and the smoothlipopolysaccharide deficient mutants described in Walker, S. G. et al.1994. “Characterization of Mutants of Caulobacter crescentus Defectivein Surface Attachment of the Paracrystalline Surface Layer”. J.Bacteriol. 176:6312-6323.

A heterologous polypeptide referred to herein may be any peptide,polypeptide, protein or a part of a protein which is desired to beexpressed in Caulobacter and which may be secreted by the bacterium. Theheterologous polypeptide includes enzymes and other functional sequencesof amino acids as well as ligands, antigens, antigenic epitopes andhaptens. The size of the heterologous polypeptide will be selecteddepending upon whether an intact S-layer is to be produced in theCaulobacter or whether the chimeric protein to be recovered from thebacterial medium as described below. Preferably, the cysteine content ofthe heterologous polypeptide and the capacity for formation ofdisulphide bonds within the chimeric protein will be kept to a minimumto minimize disruption of the secretion of the chimeric protein.However, the presence of cysteine residues capable of forming adisulphide bond which are relatively close together, may not affectsecretion.

Once a particular bacterium's S-layer protein gene is characterized,this invention may be practised by implementing one or more knownmethods to insert a selected heterologous coding sequence into all orpart of the S-layer protein gene so that both the S-layer protein andthe heterologous sequence are transcribed “in-frame”. Knowledge of anS-layer protein gene sequence permits one to identify potential sites toinstall the heterologous genetic material. The repetitive nature of theprotein in the S-layer permits multiple copies of a heterologouspolypeptide to be presented on the surface of the cell.

The following general procedure lays out courses of action and specifiesparticular plasmid vectors or constructions that may be used toaccomplish fusion of an S-Layer protein with a polypeptide of interest.The following description uses the rsaA (S-layer) gene of C. crescentusas an example (see FIG. 6 and SEQ ID NO:6). The latter gene sequence ischaracterized in Gilchrist, A. et al (1992) [supra].

The general procedure includes detailed steps allowing for the followingpossibilities:

1) use of a collection of potentially permissive sites in the S-layergene to install the genetic information for a polypeptide of interest;

2) use of a Carrier cassette for delivering a gene of interest to siteswithin the S-layer gene (the cassette offers several advantages overdirect modification of a gene of interest, in preparation forinsertion);

3) creation of a collection of random insertion sites based on arestriction enzyme of choice, if the available collection of potentiallypermissive sites is for some reason unsuitable; and,

4) preparation of DNA coding for a polypeptide of interest for directinsertion into permissive sites (ie, not using the Carrier cassette) bya method best suited for the particular case (several options aresuggested).

The general procedure involves the following steps and alternativecourses of action. As a first step the practitioner will choose anappropriate region (or specific amino acid position) of the S-layer forinsertion of a desired polypeptide. Second, the practitioner will createa unique restriction site (preferably hexameric) in the rsaA (S-layer)gene at a position within the gene encoding that region (orcorresponding to a specific amino acid) using either standard linkermutagenesis (regional) or site directed mutagenesis (specific aminoacid). The unique restriction site will act as a site for accepting DNAencoding the polypeptide of interest. The plasmid-based promoter-lessversion of the rsaA gene (pTZ18U:rsaAΔP) shown in FIG. 2 may be usedbecause it contains an appropriate combination of 5′ and 3′ restrictionsites useful for subsequent steps (see: Gilchrist, A. et al (1992)[supra]). The restriction site should not occur in rsaA, its carrierplasmid or the DNA sequence coding for the polypeptide of interest.

If it is unclear which region of the S-layer would be suitable forinsertion of a polypeptide of interest, a random linker mutagenesisapproach is used to randomly insert a unique linker-encoded restrictionsite (preferably hexameric) at various positions in the rsaA gene. Sitesfor insertion of the linker are created using an endonuclease, either ofa sequence specific nature (e.g. tetrameric recognition site restrictionenzyme) or sequence non-specific nature (e.g. Deoxyribonuclease I [DNaseI]). A particularly suitable method is the generalized selectable linkermutagenesis approach based on any desired restriction site of: Bingle,W. H., and J. Smit. 1991 “Linker Mutagenesis Using a Selectable Marker:A Method for Tagging Specific Purpose Linkers With anAntibiotic-Resistance Gene”. Biotechniques 10: 150-152. Becauseendonuclease digestion is carried out under partial digestionconditions, a library of linker insertions at different positions inrsaA is created. Partial digestion with MspI, HinPI and Aci:I can create150 potential sites for insertion of a Bam HI linker such as:

5′-CGACGGATCCGT (SEQ ID. NO:10).      TGCCTAGGCAGC-5′

If restriction endonucleases are used to create sites for subsequentinsertion of a linker encoding a hexameric restriction site, mutagenesismay also be done with a mixture of 3 different linkers incorporatingappropriate spacer nucleotides in order to satisfy reading frameconsiderations at a particular restriction site (only 1 of the 3 linkerinsertions will be useful for subsequent acceptance of DNA encoding thepolypeptide of interest). With DNase I, only one linker is needed, butagain only 1 of 3 linker insertions may be useful for accepting DNAencoding the polypeptide of interest depending on the position of theDNase I cleavage with respect to the 3 bases of each amino acid codon.

Next, a linker tagged with a marker is used to insert DNA of interest ata restriction site. For example, if BamHI sites are appropriate as sitesfor the introduction of DNA encoding a polypeptide of interest, BamHIlinkers tagged with a kanamycin-resistance gene for selectable linkermutagenesis may be used. One such 12-bp linker carried in plasmidpUC1021K was described by Bingle and Smit (1991) [Supra]. Two additional15-bp linkers (pUC7165K and pTZ6571K) constructed for creating the other2 possible translation frames within the linker insert itself aredescribed in FIGS. 3 and 4 (SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; and,SEQ ID NO:5). Any one of the above three kanamycin-resistance taggedBamHI linkers is suitable for mutagenesis at sites created in rsaA byDNase I. As outlined above, a mixture of all three linkers is preferablyused for mutagenesis at sites created in rsaA by restriction enzymedigestion.

Once a library composed of linker insertions encoding desired hexamericrestriction site at different positions in rsaA has been created, theDNA encoding a polypeptide of interest is inserted into the sites enmasse (the library of mutated rsaA genes may be manipulated as oneunit). The library is digested with the restriction enzyme specific forthe newly-introduced linker encoded restriction site and ligated to aDNA fragment encoding the polypeptide of interest and carrying theappropriate complementary cohesive termini. The DNA specifying thepolypeptide of interest can be prepared by a number of standard methods,which may include oligonucleotide synthesis of 2 anti-complementarystrands, polymerase chain reaction (PCR) procedures, or addition oflinkers whose termini are compatible with the introduced sites in rsaAto a suitably modified segment of DNA.

In order to facilitate the rapid recovery of useful rsaA genes carryingnewly inserted DNA at BamHI sites encoding the polypeptide of interest,the Carrier oligonucleotide shown in FIG. 1 may be used. The Carrier isdesigned to accept DNA (including multiple copies and mixtures) preparedby PCR or annealed synthesized oligonucleotides and controls directionof insertion of the foreign segment into a rsaA gene through use of apromoterless drug resistance marker. The DNA of interest is firstdirectionally cloned, if possible, using the XhoI, StuI, or SalI sitesor non-directionally cloned using any one of the sites in the sameorientation as a promoterless chloramphenicol resistance (CmR) gene. Todo this the DNA of interest must be provided with the appropriatetermini for cloning and spacer nucleotides for maintaining correctreading frame within the cassette and should not contain a BGlII site.For insertion into the BamHI linker library, the DNA of interest isrecovered as a BamHI fragment tagged with a CmR gene. When ligated tothe BamHI digested rsaA linker library, only those colonies of thebacterium (eg. E. coli) used for the gene modification steps that arerecovered will be those carrying insertions of the desired DNA in thecorrect orientation, since the promoter on the plasmid is 5′ to rsaAΔPand the CmR gene. This eliminates screening for DNA introduction andincreases the recovery of useful clones by 100% (1 of 3 versus 1 of 6).While still manipulating the library as one unit, the CmR gene isremoved using BglII. The carrier oligonucleotide also provides theopportunity to add DNA 5′ or 3′ to the DNA of interest at SalI, XhoI orStuI sites providing the DNA of interest does not contain any of thesesites. This allows some control over spacing between rsaA sequences andthe sequence of the DNA of interest.

Next, the rsaA genes carrying the DNA of interest in the correctorientation is excised from the plasmid (eg. from the pTZ18U:rsaAΔPplasmid) and is transferred to a suitable vector providing a promoterrecognized by Caulobacter. Such vectors include pWB9 or pWB10 (asdescribed in Bingle, W. H., and J. Smit. 1990). “High Level PlasmidExpression Vectors for Caulobacter crescentus Incorporating theTranscription and Transcription-Translation Initiation Regions of theParacrystalline Surface Layer Protein Gene”. Plasmid 24: 143-148) withEcoRI/SstI sites. The DNA of interest should not contain the samerestriction sites present in the vector. The latter vectors allowexpression of rsaA hybrids in S-layer negative mutants of Caulobactersuch as CB15KASac.

Those Caulobacter surviving transfer are examined for chimeric proteinsecretion, S-layer assembly and presentation of the new polypeptideactivity, antigenicity, etc. by methods specific to the needs of theinvestigator or the capabilities of the inserted sequence. Many of thesites created are “benign” as they have no effect on the functionalregions of the protein involved with export, self assembly, etc.However, not every site that results in an absence of functionaldisruption of the S-layer is best for insertion of new activities. Somesites may not be well exposed on the surface of the organism and othersites may not tolerate insertion of much more DNA than the linkersequence.

By selecting the site of insertion of the heterologous material, it ispossible to express heterologous polypeptides of up to about 60(preferably less than 50) amino acids in a S-layer chimeric proteinwhich will assemble as an S-layer on the cell surface. Single ormultiple insertions of smaller polypeptides (eg. 10-20 amino acids) at awide range of the permissive sites in the S-layer gene will permitS-layer formation. Some sites, as reported herein, are sensitive to evensmall insertions resulting in the chimeric protein being released intothe medium. Release may also be deliberately affected by use of ashedding strain of Caulobacter to express the chimeric protein or byphysical removal of the S-layer from whole cells.

Where S-layer formation is not required, this invention permits theexpression of quite large polypeptides (eg. about 200 amino acids) aspart of the S-layer protein. Expressing a chimeric protein containing aS-layer protein component having substantial deletions, as describedbelow, may increase the size of the heterologous polypeptides that willbe expressed and secreted by Caulobacter.

The preceding methods describe insertion of linkers in-frame into anrsaA gene (eg. a promoterless version of the gene). The sites that areintroduced allow subsequent insertion of foreign DNA in-frame into thefull length rsaA gene. This invention also includes the construction ofchimeric S-layer protein genes and the resulting production of chimericS-layer proteins wherein the S-layer gene component is highly modifiedby deleting large portions of that gene which reduces the amount ofCaulobacter protein present in the secreted chimeric protein.

Generally, large deletions throughout the S-layer gene will result in achimeric protein that is not capable of forming an S-layer. Attachmentof the S-layer to the cell is abolished if about the first 29 N-terminalamino acids of the S-layer protein are deleted. Deletion of the first776 amino acids from the N-terminal region will still result in achimeric protein that is secreted from the cell but having a S-layerprotein component of only the 250 C-terminal amino acids. It has alsobeen found that only the extreme C-terminal region corresponding toapproximately amino acids 945-1026 of RsaA is required for secretion ofan S-layer chimeric protein from Caulobacter. Thus the chimeric proteinneed only have the 82 amino acid C-terminal region of the S-layerprotein to be secreted from the cell. Furthermore, use of the C-terminalregion corresponding to about amino acids 850-1026 (or more) of RsaA notonly permits the cell to transport the chimeric protein outside of thecell, but also promotes spontaneous aggregation of much of the secretedchimeric protein in the cell medium and formation of a macroscopicprecipitate that may be collected with a course mesh or sheared tomicron-sized particles which may be ideal for vaccine presentation.Yields of up to 250 mg. (dry weight) of protein per liter of cells maybe possible.

Sequence analysis of the 3′ region of the S-layer genes from differentstrains of Caulobacter shows that the portion of the gene encoding theC-terminal region of the S-layer protein is highly conserved along withthe immediate downstream non-translated and translated region. Sequenceanalysis of the S-layer genes and downstream regions in CB15 and CB2A(which are readily distinguishable strains) shows identical DNAsequences coding for the last 118 C-terminal amino acids of the S-layerprotein and the downstream non-translated region. Sequencing of the nextdownstream translated gene to amino acid 97 of the gene product showsonly a single base pair change between CB15 and CB2A, resulting in aconservative amino acid substitution in the translation product.Conservation of the C-terminal region of Caulobacter S-layer protein andassociated coding regions shows that this invention may be carried outusing any Caulobacter producing a S-layer protein.

This invention may be practised as shown in the Examples by expressionof modified S-layer genes borne on plasmids that are broad host rangevectors capable of being expressed in Caulobacter. Such plasmids arereadily constructed and introduced to Caulobacter by electroportation.Typically, the plasmid is maintained in the Caulobacter by antibioticselection. Highly modified rsaA genes with attached heterologoussequences may also be introduced into Caulobacter on a plasmid that isnot replicated by Caulobacter. At a low but practicable frequency,homologous recombination of the incoming modified S-layer gene with thechromosome-resident copy of the S-layer gene in the cell will result ina gene rescue or transfer event. In some cases it may be desirable toobtain a stable cell line in which the chimeric S-layer gene ischromosomal. Various protocols for creating chromosomal insertions areset out in the Examples.

Use of the S-layer protein as a vehicle for production of a heterologouspolypeptide has several advantages. Firstly, the S-layer protein issynthesized in large quantities and has a generally repetitive sequence.This permits the development of systems for synthesis of a relativelylarge amount of heterologous material as a fusion product with anS-layer protein (chimeric protein). It may be desirable to retain thechimeric protein as part of the bacterial cell envelope or, the fusionproduct may be separated from the organism, such as by the methoddescribed in: Walker, S. G., et al. 1992. “Isolation and Comparison ofthe Paracrystalline Surface Layer Proteins of Freshwater Caulobacters”.J. Bacteriol. 174:1783-1792. Alternatively, the Caulobacter strain thatis used to express the fusion product may be derived from a strain suchas CB15Ca5 that sheds its S-layer.

This invention is particularly suited for use in a bioreactor systems.An example would be the use of a modified Caulobacter expressing apolypeptide having activity similar to that of a metallothionein in abioreactor, to bind toxic metals in sewage, waste water etc.Caulobacters are ideal candidates for fixed-cell bioreactors, theconstruction of which is well known. An example of such a bioreactor isa rotating biological contactor. Although other bacteria are found inthe environment that are capable of binding metals, they often do so byproducing copious polysaccharide slimes that quickly plug filtrationsystems. In some cases, the bacteria are not surface-adherent or thebacteria do not show selectivity towards key toxic metals. By takingadvantage of the natural bio-film forming characteristics ofCaulobacter, bioreactors may be formed comprising a substrate and asingle layer of cells adhered thereon, with the cells distributed athigh density. A variety of substrates may be used such as a column ofchemically derivatized glass beads or a porous ceramic material such asceramic foam.

Another advantageous application for this invention is in the productionof batch cultures of modified Caulobacter wherein the S-layer protein isa fusion product with an enzyme. For example, such Caulobacter could begrown in wood pulp suspensions at an appropriate juncture of the pulpingprocess in order to provide for enzymatic decomposition of the wood-pulpstructure (e.g. with an enzyme having an activity like xylanase orcellulase). Such an application may permit more effective penetration ofbleaching agents in the wood-pulp bleaching process thereby reducing theuse of chlorine-based bleaching agents.

Examples of enzymes that may be expressed as chimeric S-layer proteinsinclude alkaline phosphatase (eg. by expression of the pho A gene of E.coli; see: Hoffman, C. S., and Wright, A. 1985. “Fusions of SecretedProtein to Alkaline Phosphatase: An Approach for Studying ProteinSecretion”. Proc. Natl. Acad. Sci. U.S.A. 82:5107-5111; Bingle, W. H.,et al. 1993.“An “All Purpose” Cellulase Reporter for Gene Fusion Studiesand Application to the Paracrystalline Surface (S)-Layer Protein ofCaulobacter crescentus”. Can.J. Microbiol.39: 70-80; and Bingle, W. H.and Smit, J. 1994. “Alkaline Phosphatase and a Cellulase ReporterProtein Are Not Exported From the Cytoplasm When Fused to LargeN-terminal Portions of the Caulobacter crescentus Surface (S)-LayerProtein”. Can.J. Microbiol. 40:777-782.) and, cellulase (eg. byexpression of the CenA gene of Cellulomonas fimi; see: Bingle, W. H. etal. (1993) [supra]; and Bingle, W. H. and Smit, J. (1994) [supra]).

Another advantageous application of this invention is the production oforganisms that secrete and optionally present vaccine-candidateepitopes. For example, modified Caulobacter may be readily cultured inoutdoor freshwater environments and would be particularly useful in fishvaccines. The two-dimensional crystalline array of the S-protein layerof Caulobacter, which has a geometrically regular, repetitive structure,provides an ideal means for dense packing and presentation of a foreignepitope to an immune system in cases where the epitope is part of anintact S-layer in the bacterial cell surface.

This invention also provides an efficient expression system forpolypeptides that may be harvested in large quantities relatively freeof contaminants and protein of Caulobacter origin. Expression of aheterologous polypeptide fused with sufficient C-terminal amino acids ofthe S-layer protein to promote secretion of the heterologous polypeptideresults in the accumulation of large quantities of secreted protein inthe cell medium. In such cases, the chimeric protein does not have to bereleased from the cell surface. Furthermore, adjustment of the size ofthe S-layer protein portion can dictate whether the secreted chimericprotein is soluble or will precipitate in the cell medium. Thisembodiment may also be useful in cases where the Caulobacter is toexpress a foreign antigenic component and it is desired to minimize theamount of Caulobacter protein that is associated with the foreignantigen secreted by the Caulobacter.

EXAMPLE 1 Production of Permissive Insertion Sites in C. crescentus

Using the restriction enzyme TaqI, a partial digestion of the rsaA genein pTZ18U:rsaAΔP produced a group of linearized segments with randomTaqI sites cleaved. The linearized segments were modified by use of thetagged linker mutagenesis procedure of Bingle and Smit (1991) [supra],using the 12-bp BamHI linker carried in plasmid pUC102K discussed in thegeneral procedure above. Those products that produced a full-lengthprotein in E. coli were ultimately transferred to pWB1 (a minorvariation of pWB9 that is replicated by Caulobacter), as described inthe general procedure. The resulting construction was introduced into aC. crescentus strain. Distinguishable events were retrieved and analyzedfor the ability to produce a full-length protein in C. crescentus and toproduce the crystalline S-layer on their surface and the approximatelocation of the insertion. Cells were screened for the presence of aS-layer protein of approximately 100 kDa that is extracted from thesurface of whole cells by 100 mM HEPES at ph2. The results of thisscreening together with the approximate positions of five successfulevents (and subsequently determined exact or specific insertionpositions) are illustrated in FIG. 5.

The above-described five positive events represent cases where the4-amino acid insertion is tolerated with no effect on the S-layerfunction. The S-layers of the modified Caulobacter wereindistinguishable from a wild-type S-layer. Thus, they have a higherpotential for tolerating the addition of more foreign peptide materialthan less characterized sites. By producing 3 versions of the gene ofinterest, representing each possible reading frame (using standardlinker addition technology), one may test each of these sites forsuitability in expressing the desired activity. Also, by usingrestriction enzymes other than TaqI (such as AciI, HinPI or MspI) alarger library of BamHI insertions may be created.

EXAMPLE 2 Insertion of Cadmium Binding Polypeptides into Specific Sites

An insertion of the above described 12 bp linker was made at the TaqIsite that corresponds to amino acid #188, frame #3 (see FIG. 6; SEQ IDNO:6; and, SEQ ID NO:7). This created a unique BamHI site at thatposition. Because the precise position of the TaqI site could beassessed from the DNA sequence information available for the rsaA gene,the necessary translation frame was known and thus a single constructionof a metallothionein gene was made. This was done by excision of thecoding sequence of monkey metallothionein II peptide (60 amino acidscomprising 10 cysteine residues and having a molecular weight of about5000) at known restriction sites and adapting the gene ends with BamHIlinkers with appropriate base pair spacers for the needed translationframe.

After insertion into the BamHI site created at position 188, frame 3,several clones were examined by determining whether they could bindelevated levels of cadmium by the assay described below. The assay wasnecessary because the segment had equal probability of being insertedbackwards. One clone that gave positive results was examined by electronmicroscopy and the presence of a normal S-layer was confirmed. Theplasmid in the clone that gave positive results was also examined by DNAsequencing analysis, sequencing across the junction between the position188 site and the 5′ side of the metallothionein gene. The sequence dataconfirmed correct orientation.

The plasmid-containing clone and relevant control strains were examinedfor the ability to bind several metals known to be bound by nativemetallothionein. This was done by growing the strains of bacteria in thepresence of the metals at a concentration of 5 ug/ml. After extensivewashing of the cells to remove unbound metal, the cells were ashed bytreatment at 500° C. and the residue was dissolved in dilute nitric acidand examined for metal content by atomic absorption spectroscopy. Theresults from one round of data collection is shown in Table 1. In thecase of cadmium and copper, an elevated level of bound metal is noted inthe metallothionein-expressing strains.

TABLE 1 Metal Ion Tested (μg/metal/OD unit of cells Copper CaulobacterTrial 1 2 Cadmium Zinc CB15 1.79 1.0 0.71 4.15 (wild-type,S-layer[+])CB15KSAC 2.18 1.33 1.07 4.09 (S-layer negative strain) CB15KSAC/p188.32.01 1.30 11.1 3.66 (contains S-layer with linker insert only)CB15KSAC/p188.3MT 2.79 3.09 19.1 3.00 (S-layer with Metallothioneininserted)

EXAMPLE 3 Investigation of Other Permissive Sites in rsaA Gene

A library of 240 BamHI linker insertions was created using theprocedures of Example 1. of the 240 insertions, 45 target sites in thersaA gene were made with TaqI. 34 of the latter insertions werediscarded because the clones contained deletions of rsaA DNA as well asthe linker insertions. The remaining 11 resulted in 5 non-permissive andthe 6 permissive sites described in Example 1. The remaining 195insertions in the library were made using the enzymes HinPI, AciI, andMspI to create target sites as outlined in Example 1. Of the latter 195insertions, 49 permissive sites were located for a total of 55. Of thosesites scored as non-permissive, some may have had deletions of rsaA DNAat the linker insertion site. One BamHI linker insertion at a TaqI sitethought to be permissive was later found by nucleotide sequencing to belocated outside the rsaA structural gene reducing the total number ofpermissive sites to 54 from 55.

FIG. 7 illustrates the approximate location by restriction mapping of 54permissive sites. The results show that sites that will accept 2-4 aminoacids while still allowing the protein to be made and assembled into anS-layer are scattered up and down the protein. Furthermore, there is anunexpectedly high proportion of sites at which such insertions do notprevent expression and assembly of the S-layer. The results indicatethat approximately 25-50% of in-frame linker insertions will betolerated by the S-layer protein and the Caulobacter and that diverseregions of the protein will tolerate insertions. Thus, Caulobacter is anideal candidate for expression of polypeptides fused with the S-layerand the presence of multiple permissive sites extending along the rsaAgene will permit the insertion of a plurality of the same or differentpeptides into the same RsaA protein molecule and expressed on thesurface of a single Caulobacter.

EXAMPLE 4 Further Studies with Cadmium Binding Polypeptides

The results described for Example 3 indicated that it would be possibleto insert metallothionein at multiple places in the RsaA protein andthereby enhance the metal binding capacity of such a transformedCaulobacter. However, when the procedures of Example 2 were repeated toinsert the metallothionein coding sequence into others of the 54permissive sites identified in the preceding Example in each case, thetransformed Caulobacter did not secrete a chimeric protein and did notsynthesize an S-layer. Furthermore, the transformed Caulobacter ofExample 2 was stable as long as the transformants were frozenimmediately after isolation. When continuously cultured forapproximately one week, the transformants deleted the metallothioneinportion of the S-layer and the S-layer protein returns to its normalsize.

Consideration of the predicted amino acid sequence of the rsaA proteinshows that the latter protein lacks cysteine residues whereasmetallothionein has a high cysteine content. It thus appeared that forsecretion and long term expression of a RsaA chimeric protein, theheterologous polypeptide portions of the chimeric protein should nothave high cysteine content and preferably, not be capable of formingmultiple disulphide bonds in the chimeric protein in an aerobicenvironment.

Following the foregoing procedures, single and multiple copies of DNAencoding the synthetic cadmium binding peptide shown in FIG. 10 (SEQ IDNO:11) was synthesized, inserted at the amino acid 277 site of rsaAusing the above described Carrier cassette and was expressed in C.crescentus. The peptide has a single cysteine residue. Mild acidextracts of whole cells expressing the modified rsaA gene were subjectedto SDS-PAGE for identification of S-layer proteins. The S-layer proteinwas expressed and secreted when there was from 1 to 3 copies of thecadmium binding peptide present at RsaA amino acid position 277.Insertion of 4 or more copies resulted in a dramatic reduction ofS-layer protein released from the whole cells by mild acid treatment tobarely detectable levels. Detection by autoradiography of RsaA proteinin vivo labelled with 35 S-cysteine and in vitro with 125I-iodoacetamide confirmed that the cadmium binding peptide was part ofthe chimeric RsaA protein. This demonstrates that Caulobacter crescentusis capable of secretion of a chimeric rsaA protein having a limitedcysteine content and a limited capacity for disulphide bond formationwithin the chimeric protein.

EXAMPLE 5 Expression and Presentation of Antigenic Epitopes onCaulobacter Cell Surface

Using the library of the 49 permissive sites other than those made withTaqI described in Example 3, the coding sequence for the 12-amino acidpilus peptide epitope lacking cysteine residues from Pseudomonasaeruginosa PAK pilin was inserted at the sites using the proceduresdescribed above and employing the Carrier cassette shown in FIG. 1.Positioning of the added DNA between the first Bam HI site and the BglII site permitted use of the latter site for making repeated insertionsof DNA. The coding sequence for the peptide shown in FIG. 8, includingboth cysteine residues was also inserted in separate experiments.

DNA coding for the peptide shown in FIG. 8 (SEQ ID NO:8) was prepared byoligonucleotide synthesis of two anti-complementary strands. Thetransformed bacteria were screened for both production and presentationof the epitopes by the transformed Caulobacter by using standard Westernimmunoblot analysis (see: Burnette, W. N. 1981. “Western Blotting;Electrophoretic Transfer of Protein from Sodium Dodecyl-PolyacrylamideGels to Unmodified Nitrocellulose and Radiographic Detection Antibodyand Radioiodinated Protein A”. Analytical Biochemistry 112:195-203) andby colony immunoblot tests in which the cells were not disrupted (see:Engleberg, N. C., et al. 1984. “Cloning an Expression of Legionellapneumophilia Antigens in Escherichia coli”. Infection and Immunity44:222-227). Anti-pilus monoclonal antibody obtained from Dr. Irvin,Dept. of Microbiology, University of Alberta (Canada) was used in theimmunoblot analyses to detect the presence of the pilus epitope insert.The antibody (called PK99H) was prepared using purified Pseudomonasaeruginosa PAK pilin as the antigen and the monoclonal antibody againstthe 12 amino acid epitope was isolated by standard techniques usingBALB/C mice as a source of ascites fluid. Reaction with the antibody inthe whole cell colony immunoblot assay shows that the epitope is notonly expressed in the transformed Caulobacter but is exposed on theS-layer surface overlying the cell in such a way that the epitope isavailable to the antibody. When the two cysteine residues of the pilinepitope were incorporated in the chimeric protein, the protein was stillexpressed and secreted at normal levels.

Of the organisms screened, insertions of the pilus epitope at thefollowing sites in the rsaA gene as determined by nucleotide sequencingresulted in a positive reaction with the antibody in the whole cellColony immunoblot analysis: 69, 277, 353, 450, 485, 467, 551, 574, 622,690, 723, and 944. The results show that the permissive sites that willaccept polypeptides of the size of the pilus epitope are numerous andscattered across the rsaA gene.

Further studies with the pilus peptide resulted in successful expressionand secretion of RsaA chimeric proteins have single copies of thepeptide at the locations shown in FIG. 11. Also, four and seven copiesof the 12 amino acid pilus peptide were expressed and secreted as a RsaAchimeric protein when inserted at amino acids 277 and 551 respectivelyof the RsaA protein. However, insertions of the pilus peptide at aminoacids 69, 277, 450, 551 and 622 resulted in a chimeric protein that didnot attach to the cell surface and was released into the culture medium.

EXAMPLE 6 Insertion of Large Polypeptides

Bacterial surface proteins from organisms other than Caulobacterdescribed in the prior art are generally not known to acceptpolypeptides larger than about 60 amino acids within the structure ofthe surface protein. The procedures of the preceding Example werecarried out in order to insert the coding sequence of a 109 amino acidepitope from IHNV virus coat glycoprotein at insertion sites identifiedin the preceding Example. The IHNV epitope was prepared by PCR and hadthe portion of the sequence shown in FIG. 9 (SEQ ID NO:9) which isequivalent to amino acid residues 336-444 of the IHNV sequence describedin: Koener, J. F. et al. 1987. “Nucleotide Sequence of a cDNA CloneCarrying the Glycoprotein Gene of Infectious Hematopoietic NecrosisVirus, a Fish Rhabdovirus”. Journal of Virology 61:1342-1349. Anti-IHNVpolyclonal antibody against whole IHNV obtained from Dr. Joann Leong,Dept. of Microbiology, Oregon State University, U.S.A. (see: Xu, L. etal. 1991. “Epitope Mapping and Characterization of the InfectiousHematopoietic Necrosis Virus Glycoprotein, Using Fusion ProteinsSynthesized in Escherichia coli”. Journal of Virology 65:1611-1615) wasused in the immunoblot assays described in the preceding Example toscreen for Caulobacter that express and present the IHNV sequence on thesurface of the S-layer of the Caulobacter. Reaction in the whole cellcolony immunoblot assay was positive in respect of insertions at sites450 and 551, and negative at a site which was at approximately aminoacid 585.

The IHNV insert contains a single cysteine residue and is an extremelylarge insert for successful expression as a fusion product with abacterial surface protein.

In further studies, the same 109 amino acid portion of the IHNVglycoprotein was inserted at amino acid 450 of the RsaA protein. Thechimeric protein expressed and secreted by Caulobacter crescentus andwas recovered from the cell culture medium. SDS-PAGE analysis of therecovered proteins showed that some of the chimeric proteins weresmaller than the predicted rsaA chimeric protein but still boundanti-IHNV antibody. Analysis of these proteolytic products showed thatcleavage of the chimeric protein occurred at an Arg residue encoded bythe gene transfer cassette shown in FIG. 1. Thus in some cases,adjustment of the nucleotide sequence at the interface of thepolypeptide and rsaA coding sequences may be necessary to preventexpression of an arginine residue.

EXAMPLE 7

Methods are described above for the insertion of 12-bp BamHI linkersites into a promoterless version of the rsaA gene. Because linkerinsertions involve the insertion of 12 bp (i.e. a multiple of three) anin-frame linker insertion resulted in every case. These linker sites areintroduced to allow subsequent insertion of DNA encoding foreignpeptide/proteins. Expression of such chimeric genes leads to theproduction of an entire full-length RsaA protein carrying the insertedheterologous amino acid sequence of interest. A number of BamHI sitepositions were identified above precisely by nucleotide sequencing. Fourof the sites in the rsaA gene correspond to amino acid positions 188,782, 905, 944 in the RsaA protein. For this example, an additionallinker insertion was created at amino acid position 95 of the nativegene (i.e. this gene carried its own promoter) using the samemethodology. All five in-frame BamHI linker insertion sites wereinserted in the rsaA so that the nucleotides of the linker DNA were readin the reading frame GGA/TCC (FIG. 12).

Because all BamHI linker nucleotides were read in the same readingframe, the 5′ region of one rsaA gene carrying a BamHI linker insertionat one position could be combined with the 3′ region of an rsaA genecarrying another of the BamHI linker insertions to create in-framedeletions with a BamHI site at the joint between adjacent regions ofrsaA. Using such a method, in-frame deletions of rsaA (ΔAA95-782) andrsaA(ΔAA188-782) were created.

DNA fragments encoding various C-terminal portions of the 1026 aminoacid RsaA protein were isolated using the newly inserted BamHI linkersites as the 5′-terminus of the fragment and a HindIII site as the 3′terminus of the fragment. These BamHI fragments were transferred to theBamHI/HindIII sites of pUC8 (J. Vieira, and J. Messing. 1982. “The pUCPlasmids, an M13mp7-Derived System for Insertion Mutagenesis andSequencing With Synthetic Universal Primers” Gene 19:259-268) creating“rsaA C-terminal Segment Carrier plasmids” (FIG. 12). The insertion intopUC8 also resulted in the creation of an in-frame fusion between thefirst 10 N-terminal amino acids of LacZa and the various C-terminalfragments (AA782-1026, AA905-1026 or AA944-1026) of RsaA. TheseLacZa:rsaA fusion proteins can be produced in C. crescentus using thelacZa transcription/translation initiation signals when introduced onappropriate plasmid vectors or direct insertion into the chromosome(see: W. H. Bingle, et al. 1993. “An All-Purpose Cellulase Reporter forGene Fusion Studies and Application to the Paracrystalline Surface(S)—Layer Protein of Caulobacter crescentus.” Can. J. Microbiol.39:70-80).

Both types of constructions (i.e., the deletion versions and theC-terminal only segments) result in the production of proteins that aresecreted in Caulobacter strains as highly modified RsaA proteins. Thegene segments can also facilitate the secretion of heterologouspolypeptides by insertion or fusion of appropriate DNA sequences at theunique BamHI site that exists in each of the constructions. Thefollowing describes specific methods for doing so to create chimericproteins capable of secretion in C. crescentus.

A—Creating Fusions of Desired Sequences with C-terminal Portions ofrsaA—Method 1

The general process is as follows:

1) Inserting the desired sequence into the Carrier cassette. Thefollowing describes the specific manner in which heterologous sequencesmay be introduced into the Carrier cassette of FIG. 1.

a) Insertion of a single copy of the desired gene segment.

Depending upon the length of the gene segment, two methods ofconstruction may be used. For segments of up to about 30 amino acids,two oligonucleotides of appropriate sequence are chemically synthesized,annealed by mixing, heating and slow cooling and then ligated into theCarrier cassette. The oligonucleotides will also contain additional basepairs that recreate “sticky ends” of appropriate restrictionendonuclease sites at each end of the duplex DNA that results from theannealing process.

For longer segments, PCR is used to amplify a region of a target DNAsequence. Oligonucleotides are synthesized that have sequencecomplementary to the boundaries of the desired sequence and whichcontain additional base pairs that recreate a “sticky end” of anappropriate restriction endonuclease site. In the present exampleoligonucleotides are made to produce products with the appropriaterestriction endonuclease site for directional cloning into the Carriercassette. PCR amplification of the desired sequence is then done bystandard methods.

For both methods, the sticky ends prepared must be appropriate for anXhoI site at the 5′ terminus of the desired DNA sequence and StuI orSalI sites at the 3′ terminus; this places the desired gene segment inthe correct orientation within the Carrier cassette. Reading framecontinuity is maintained by appropriate design of the oligonucleotidesused for the PCR step.

b) Preparation of multiple copies of the desired gene segment.

The Carrier cassette also allows production of multiple insert copies. ABglII site in the cassette is restored after removal of the promoterlessantibiotic resistance gene; that site can be used to insert anadditional copy of the Carrier/desired sequence insertion, using theterminal BamHI sites, because the “sticky ends” produced by both BamHIand BglII are the same. This “piggy-back” insertion still maintains thecorrect reading frame throughout the construction. Any number ofadditional cycles of “piggy-backing” can be done because the BamHI/BglIIligation results in sequence which is no longer a substrate for eitherenzyme. The result is the production of cassettes of multiple copies ofthe desired sequence which can be transferred to appropriately modifiedS-layer protein genes with the same ease as a single copy. An additionalfeature of this method is that different heterologous sequences can bepaired together in this multiple copy cassette with the same ease asmultiple copies of the same heterologous sequence.

EXAMPLE 7a

Insertion of an 109 amino acid segment of the IHNV surface glycoproteinto Carrier cassette.

Using the methods described, a PCR product was made that contained theDNA coding for amino acids 336 to 444 (FIG. 9) of the major surfaceglycoprotein of the Infectious Hematopoietic Necrosis Virus (IHNV),which infects Salmonid fish.

EXAMPLE 7b

Insertion of an 184 amino acid segment of the IHNV surface glycoproteinto Carrier cassette.

Using the methods described a PCR product was made that contained theDNA coding for amino acids 270 to 453 of the IHNV glycoprotein segmentshown in FIG. 9.

EXAMPLE 7c

Insertion of single and multiple copies and an epitope of thePseudomonas aeruginosa PAK pilus gene to Carrier cassette.

Oligonucleotides were constructed to code for the pilus epitopedescribed in Example 5, which corresponds to a sequence at the extremeC-terminus of the pilus protein. Using the methods outlined in partA(1)(b) of this Example, 3 tandem copies were prepared.

2) Transfer of Carrier cassette to the rsaA C-terminal Segment Carrierplasmids. The constructions described in examples 7a and 7b above arethen transferred to the rsaA C-terminal Segment Carrier plasmids,described above, resulting in an in-frame fusion of: a) a very shortsection of the betagalactosidase protein (10 amino acids), b) thedesired sequence flanked by 2-3 amino acids derived from Carriercassette sequence and c) the appropriate rsaA C-terminal segment. Insome cases, the first codon of the rsaA C-terminal segment is convertedto a different codon as a result of the fusion. For example, while thersaa C-terminal segment may have coded for amino acids 944-1026 of RsaA,the resulting chimeric protein may only have amino acids 945-1026 nativeto RsaA.

EXAMPLE 7d

Fusion of Carrier/109 AA and 184 IHNV segments to C-terminal rsaAsegment AA782-1026.

This was done using the Carrier cassettes described in Examples 7a and7b above and the AA782-1026 rsaA C-terminal Segment Carrier plasmiddescribed above.

EXAMPLE 7e

Fusion of Carrier/109 AA and 184 AA IHNV segments to C-terminal rsaAsegment AA905-1026.

This was done using the Carrier cassettes described in Examples 7a and7b above and the AA905-1026 rsaA C-terminal Segment Carrier plasmiddescribed above.

EXAMPLE 7f

Fusion of Carrier/109 AA and 184 AA IHNV segments to C-terminal rsaAsegment AA944-1026.

This was done using the Carrier cassettes described in Examples 7a and7b above and the AA944-1026 rsaA C-terminal Segment Carrier plasmiddescribed above.

EXAMPLE 7g

Fusion of Carrier/3x Pilus Epitope segment to C-terminal rsaA segmentAA782-1026.

This was done using the Carrier cassettes described in Example 7c aboveand the AA782-1026 rsaA C-terminal Segment Carrier plasmid describedabove.

3) Expression of the desired fusion in an appropriate Caulobacter hoststrain.

a) Plasmid-based expression.

To create plasmid vectors that can be introduced and maintained inappropriate Caulobacter strains, the entire rsaA C-terminal SegmentCarrier plasmids were fused to broad host range vectors pKT215 or pKT210(see: M. Bagdasarian, et al. 1981. “Specific-Purpose Cloning Vectors.II. Broad-Host-Range, High Copy Number RSF1010-Derived Vectors, and aHost-Vector System for Gene Cloning in Pseudomonas.” Gene 16:237-247)using the unique HindIII restriction site present in each plasmid. Theresulting plasmid is introduced into Caulobacter by conjugation orelectroporation methods and is maintained by appropriate antibioticselection.

The fusions described in examples 7d-7g were expressed in Caulobacter.In each case expression and secretion of the chimeric S-layer proteinwas detected by Western immunoblot analysis of electrophoretic gels ofthe cell culture supermutant employing the monoclonal antibody for eachof the polypeptide epitopes. The transporter signal for secretion fromCaulobacter must be in the C-terminal region of amino acids 945-1026 ofthe S-layer protein as all chimeric proteins in the examples weresecreted. Precipitation of the chimeric protein occurred with the use ofrsaA segment AA782-1026 but not AA944-1026. Recovery of precipitateusing AA905-1026 was reduced as compared to AA782-1026.

b) Selection of appropriate Caulobacter host strains.

In nearly all cases the use of a S-layer negative host strain isappropriate. C. crescentus strain CB2A and strain CB15aKSac fulfil thisrequirement. If it is important to ensure that all fusion protein is nolonger attached to the cell surface, the use C. crescentus strainsCB15SCaSKSac or CB15Ca10KSac are appropriate. These strains haveadditional mutations that result in the loss of the production of aspecific species of surface lipopolysaccharide that has beendemonstrated to be involved with the surface attachment of nativeS-layer protein as a 2-dimensional crystalline array (see: Walker S. G.et al 1994. “Characterization of Mutants of C. crescentus Defective inSurface Attachment of the Paracrystalline Surface Layer”. J. Bacteriol.176:6312-6323). Most often with the highly modified versions of theS-layer gene, this precaution is not necessary since virtually allregions of the gene that may have a role in the attachment process havebeen removed.

There are two types of growth media well suited to both propagation ofCaulobacter for general purposes, including cloning steps, and also toproduce the secreted and aggregated chimeric proteins. Example of thetwo types are: 1) PYE medium, a peptone and yeast extract based mediumdescribed in Walker et al, (1994) [supra], and 2) M6HiGG medium, adefined medium described in: Smit, J., et al 1981. “Caulobactercrescentus Pilin: Purification, Chemical Characterization andAmino-Terminal Amino Acid Sequence of a Structural Protein RegulatedDuring Development”. J. Biol. Chem. 256, 3092-3097. The latter medium isespecially appropriate for preparation of the aggregated chimericproteins since it permits growth to higher densities (thereforemaximizing protein yield) and results in purer aggregated proteins sincethere are no medium derived proteins to contaminate the chimericproteins retrieved.

B—Creating Fusions of Desired Sequences with C-terminal Portions ofrsaA—Method 2.

Methods other than the use of the Carrier cassette plasmids are possibleto create heterologous insertions into deletion versions of a S-layergene or to create fusions with C-terminal portions of the S-layerprotein. PCR may be used although other known methods may also be used.The general procedure is as follows:

1) Use PCR to prepare appropriate segments:

a) Preparation of amplified segment with appropriate ends is carried outin a manner similar to that described part A(1)(a) above.Oligonucleotides are designed and synthesized such that they will annealto appropriate regions of the desired heterologous DNA and also contain“sticky ends” of appropriate sequence and frame so that the resultingPCR product can be directed inserted into appropriate modified S-layergenes.

b) Transfer to appropriate C-terminal rsaA segments is carried out byinserting the PCR products into the C-terminal segments AA782-1026,AA905-1026, or AA944-1026, as described in Examples 7d-7g above. Inaddition to the BamHI site described, the EcoR1 restriction site couldalso be used as the 5′ terminus of the incoming PCR segment, since thissite is also available in the pUC8 vector and not in the S-layer gene,so long as the correct reading frame was maintained when designing theoligonucleotides used to prepare the PCR product.

2) Expression of the desired fusion in an appropriate Caulobacter hoststrain is carried out using the procedures outlined in part A(3) above.

C—Creating Insertions of Desired Sequences into Versions of a S-layerGene Having Large Internal In-frame Deletions.

The general process is as follows:

1) Creating appropriate in-frame deletions.

rsaA (ΔAA95-782) and rsaA(ΔAA188-782) were prepared as described above.Because most of the BamH1 linker insertion sites are in the same readingframe with respect to each other, it is possible to combine other pairsof 5′ and 3′ segments using the same general method, with the sameresult of maintenance of correct reading frame throughout. Thesedeletion versions must then be tested individually to ensure thatS-layer protein is still secreted by the Caulobacter.

2) Insertion of a Gene Segment Carrier cassette containing is thedesired sequences: as described at part A(1) above, carried out usingthe procedure described in part A(2) above.

EXAMPLE 7h

Insertion of the 109 AA IHNV segment into rsaA (ΔAA95-782) and insertionof the 109 AA IHNV segment into rsaA(ΔAA188-782) is carried out as inExamples 7d-7g above. Expression of the desired genetic construction inappropriate C. crescentus strains is done using the procedures outlinedin part A(3) above.

3) Alternate PCR procedures: can be used to prepare a heterologoussegment for direct insertion into the BamHI site with the deletionversions of the rsaA gene. The procedure is essentially the same asdescribed in part B(1) above.

EXAMPLE 8 (Transfer to the native S-layer gene chromosomal site as asingle crossover event).

The fusion of the Carrier cassette with appropriate heterologous DNAsegments to a C-terminal S-layer protein segment plasmid results in apUC8-based plasmid that is not maintained in Caulobacter. Selection forthe antibiotic marker on the plasmid results in detection of the rescueevents. Most commonly these are single crossover homologousrecombination events. The result is a direct insertion of the entireplasmid into the chromosome. Thus the resident copy of the S-layer generemains unchanged as well as the incoming highly modified S-layer gene.In such cases it may be desirable to use Caulobacter strains in whichthe resident S-layer gene is inactivated in known ways. One example isthe use of C. crescentus strain CB15AKSac; this strain has an antibioticresistance gene cassette introduced at a position in the S-layer geneabout 25% of the way from the 5′ terminus.

EXAMPLE 9 (Transfer to the native S-layer gene chromosomal site as adouble crossover event).

In certain cases it may be desirable to completely exchange the residentS-layer gene copy with the incoming highly modified version. One methodis the incorporation of a sacB gene cassette (Hynes, M. F., et al. 1989.“Direct Selection for Curing and Deletion of Rhizobium Plasmids UsingTransposons Carrying the Bacillus subtilis sacB Gene.” Gene 78: 111-119)into the pUC8 based plasmids carrying the desired chimeric geneconstruction. This cassette contains a levansucrase gene from Bacillussubtilis that, in the presence of sucrose, is thought to result in theproduction of a sugar polymer that is toxic to most bacteria whenexpressed inside the cell. One first selects for the single crossoverevent as described in Example 8. Subsequent growth on sucrose-containingmedium results in the death of all cells except those that lose theoffending sacB gene by homologous recombination within the 2 adjacentgene copies. Two events are possible; restoration of the resident copyof the S-layer gene or replacement of the resident copy with theincoming modified gene (the latter is the desired event). A screen withinsertion DNA as probe or antibody specific to the heterologous geneproduct identifies successful gene replacement events. The methodrequires that the S-layer gene sequence or native sequence immediatelyadjacent to the S-layer gene be on both sides of the heterologoussequence (ie, Carrier cassette sequence plus heterologous DNA) and inthe present case is best suited for the deletion versions of the S-layergene.

Other methods are available for the delivery of genes to the chromosomeof a Caulobacter. Methods involving the use of the transposons Tn5 andTn7 as a means of delivery of genes to random chromosome locations areavailable (see: Barry, G. F. 1988 “A Broad-Host-Range Shuttle System forGene Insertion into the Chromosomes of Gram-Negative Bacteria.” Gene71:75-84.). The use of the xylose utilization operon as a target forchromosome insertion have also been described. This method involves theincorporation of a portion that operon into the pUC8 based plasmidconstructions described above. This allows homologous recombinationwithin the xylose operon as a means of plasmid rescue. Loss of theability to use xylose as a nutrition source is used as the means ofconfirming the rescue event.

This invention now being described, it will be apparent to one ofordinary skill in the art that changes and modifications can be madethereto without departing from the spirit or scope of the appendedclaims.

12 1 44 DNA Artificial Sequence Synthetically generated cloning site 1acgtcctagg cgagctccag ctggctccgg aggtctagac ctag 44 2 13 DNA ArtificialSequence Synthetically generated linker 2 gtcgacggga tcc 13 3 14 DNAArtificial Sequence Synthetically generated linker 3 ggatccgcgt cgac 144 14 DNA Artificial Sequence Synthetically generated linker 4 gtcgacgcggatcc 14 5 13 DNA Artificial Sequence Synthetically generated linker 5ggatcccgtc gac 13 6 3300 DNA Caulobacter crescentus CDS (101)...(3178) 6gctattgtcg acgtatgacg tttgctctat agccatcgct gctcccatgc gcgccactcg 60gtcgcagggg gtgtgggatt ttttttggga gacaatcctc atg gcc tat acg acg 115 MetAla Tyr Thr Thr 1 5 gcc cag ttg gtg act gcg tac acc aac gcc aac ctc ggcaag gcg cct 163 Ala Gln Leu Val Thr Ala Tyr Thr Asn Ala Asn Leu Gly LysAla Pro 10 15 20 gac gcc gcc acc acg ctg acg ctc gac gcg tac gcg act caaacc cag 211 Asp Ala Ala Thr Thr Leu Thr Leu Asp Ala Tyr Ala Thr Gln ThrGln 25 30 35 acg ggc ggc ctc tcg gac gcc gct gcg ctg acc aac acc ctg aagctg 259 Thr Gly Gly Leu Ser Asp Ala Ala Ala Leu Thr Asn Thr Leu Lys Leu40 45 50 gtc aac agc acg acg gct gtt gcc atc cag acc tac cag ttc ttc acc307 Val Asn Ser Thr Thr Ala Val Ala Ile Gln Thr Tyr Gln Phe Phe Thr 5560 65 ggc gtt gcc ccg tcg gcc gct ggt ctg gac ttc ctg gtc gac tcg acc355 Gly Val Ala Pro Ser Ala Ala Gly Leu Asp Phe Leu Val Asp Ser Thr 7075 80 85 acc aac acc aac gac ctg aac gac gcg tac tac tcg aag ttc gct cag403 Thr Asn Thr Asn Asp Leu Asn Asp Ala Tyr Tyr Ser Lys Phe Ala Gln 9095 100 gaa aac cgc ttc atc aac ttc tcg atc aac ctg gcc acg ggc gcc ggc451 Glu Asn Arg Phe Ile Asn Phe Ser Ile Asn Leu Ala Thr Gly Ala Gly 105110 115 gcc ggc gcg acg gct ttc gcc gcc gcc tac acg ggc gtt tcg tac gcc499 Ala Gly Ala Thr Ala Phe Ala Ala Ala Tyr Thr Gly Val Ser Tyr Ala 120125 130 cag acg gtc gcc acc gcc tat gac aag atc atc ggc aac gcc gtc gcg547 Gln Thr Val Ala Thr Ala Tyr Asp Lys Ile Ile Gly Asn Ala Val Ala 135140 145 acc gcc gct ggc gtc gac gtc gcg gcc gcc gtg gct ttc ctg agc cgc595 Thr Ala Ala Gly Val Asp Val Ala Ala Ala Val Ala Phe Leu Ser Arg 150155 160 165 cag gcc aac atc gac tac ctg acc gcc ttc gtg cgc gcc aac acgccg 643 Gln Ala Asn Ile Asp Tyr Leu Thr Ala Phe Val Arg Ala Asn Thr Pro170 175 180 ttc acg gcc gct gcc gac atc gat ctg gcc gtc aag gcc gcc ctgatc 691 Phe Thr Ala Ala Ala Asp Ile Asp Leu Ala Val Lys Ala Ala Leu Ile185 190 195 ggc acc atc ctg aac gcc gcc acg gtg tcg ggc atc ggt ggt tacgcg 739 Gly Thr Ile Leu Asn Ala Ala Thr Val Ser Gly Ile Gly Gly Tyr Ala200 205 210 acc gcc acg gcc gcg atg atc aac gac ctg tcg gac ggc gcc ctgtcg 787 Thr Ala Thr Ala Ala Met Ile Asn Asp Leu Ser Asp Gly Ala Leu Ser215 220 225 acc gac aac gcg gct ggc gtg aac ctg ttc acc gcc tat ccg tcgtcg 835 Thr Asp Asn Ala Ala Gly Val Asn Leu Phe Thr Ala Tyr Pro Ser Ser230 235 240 245 ggc gtg tcg ggt tcg acc ctc tcg ctg acc acc ggc acc gacacc ctg 883 Gly Val Ser Gly Ser Thr Leu Ser Leu Thr Thr Gly Thr Asp ThrLeu 250 255 260 acg ggc acc gcc aac aac gac acg ttc gtt gcg ggt gaa gtcgcc ggc 931 Thr Gly Thr Ala Asn Asn Asp Thr Phe Val Ala Gly Glu Val AlaGly 265 270 275 gct gcg acc ctg acc gtt ggc gac acc ctg agc ggc ggt gctggc acc 979 Ala Ala Thr Leu Thr Val Gly Asp Thr Leu Ser Gly Gly Ala GlyThr 280 285 290 gac gtc ctg aac tgg gtg caa gct gct gcg gtt acg gct ctgccg acc 1027 Asp Val Leu Asn Trp Val Gln Ala Ala Ala Val Thr Ala Leu ProThr 295 300 305 ggc gtg acg atc tcg ggc atc gaa acg atg aac gtg acg tcgggc gct 1075 Gly Val Thr Ile Ser Gly Ile Glu Thr Met Asn Val Thr Ser GlyAla 310 315 320 325 gcg atc acc ctg aac acg tct tcg ggc gtg acg ggt ctgacc gcc ctg 1123 Ala Ile Thr Leu Asn Thr Ser Ser Gly Val Thr Gly Leu ThrAla Leu 330 335 340 aac acc aac acc agc ggc gcg gct caa acc gtc acc gccggc gct ggc 1171 Asn Thr Asn Thr Ser Gly Ala Ala Gln Thr Val Thr Ala GlyAla Gly 345 350 355 cag aac ctg acc gcc acg acc gcc gct caa gcc gcg aacaac gtc gcc 1219 Gln Asn Leu Thr Ala Thr Thr Ala Ala Gln Ala Ala Asn AsnVal Ala 360 365 370 gtc gac ggg cgc gcc aac gtc acc gtc gcc tcg acg ggcgtg acc tcg 1267 Val Asp Gly Arg Ala Asn Val Thr Val Ala Ser Thr Gly ValThr Ser 375 380 385 ggc acg acc acg gtc ggc gcc aac tcg gcc gct tcg ggcacc gtg tcg 1315 Gly Thr Thr Thr Val Gly Ala Asn Ser Ala Ala Ser Gly ThrVal Ser 390 395 400 405 gtg agc gtc gcg aac tcg agc acg acc acc acg ggcgct atc gcc gtg 1363 Val Ser Val Ala Asn Ser Ser Thr Thr Thr Thr Gly AlaIle Ala Val 410 415 420 acc ggt ggt acg gcc gtg acc gtg gct caa acg gccggc aac gcc gtg 1411 Thr Gly Gly Thr Ala Val Thr Val Ala Gln Thr Ala GlyAsn Ala Val 425 430 435 aac acc acg ttg acg caa gcc gac gtg acc gtg accggt aac tcc agc 1459 Asn Thr Thr Leu Thr Gln Ala Asp Val Thr Val Thr GlyAsn Ser Ser 440 445 450 acc acg gcc gtg acg gtc acc caa acc gcc gcc gccacc gcc ggc gct 1507 Thr Thr Ala Val Thr Val Thr Gln Thr Ala Ala Ala ThrAla Gly Ala 455 460 465 acg gtc gcc ggt cgc gtc aac ggc gct gtg acg atcacc gac tct gcc 1555 Thr Val Ala Gly Arg Val Asn Gly Ala Val Thr Ile ThrAsp Ser Ala 470 475 480 485 gcc gcc tcg gcc acg acc gcc ggc aag atc gccacg gtc acc ctg ggc 1603 Ala Ala Ser Ala Thr Thr Ala Gly Lys Ile Ala ThrVal Thr Leu Gly 490 495 500 agc ttc ggc gcc gcc acg atc gac tcg agc gctctg acg acc gtc aac 1651 Ser Phe Gly Ala Ala Thr Ile Asp Ser Ser Ala LeuThr Thr Val Asn 505 510 515 ctg tcg ggc acg ggc acc tcg ctc ggc atc ggccgc ggc gct ctg acc 1699 Leu Ser Gly Thr Gly Thr Ser Leu Gly Ile Gly ArgGly Ala Leu Thr 520 525 530 gcc acg ccg acc gcc aac acc ctg acc ctg aacgtc aat ggt ctg acg 1747 Ala Thr Pro Thr Ala Asn Thr Leu Thr Leu Asn ValAsn Gly Leu Thr 535 540 545 acg acc ggc gcg atc acg gac tcg gaa gcg gctgct gac gat ggt ttc 1795 Thr Thr Gly Ala Ile Thr Asp Ser Glu Ala Ala AlaAsp Asp Gly Phe 550 555 560 565 acc acc atc aac atc gct ggt tcg acc gcctct tcg acg atc gcc agc 1843 Thr Thr Ile Asn Ile Ala Gly Ser Thr Ala SerSer Thr Ile Ala Ser 570 575 580 ctg gtg gcc gcc gac gcg acg acc ctg aacatc tcg ggc gac gct cgc 1891 Leu Val Ala Ala Asp Ala Thr Thr Leu Asn IleSer Gly Asp Ala Arg 585 590 595 gtc acg atc acc tcg cac acc gct gcc gccctg acg ggc atc acg gtg 1939 Val Thr Ile Thr Ser His Thr Ala Ala Ala LeuThr Gly Ile Thr Val 600 605 610 acc aac agc gtt ggt gcg acc ctc ggc gccgaa ctg gcg acc ggt ctg 1987 Thr Asn Ser Val Gly Ala Thr Leu Gly Ala GluLeu Ala Thr Gly Leu 615 620 625 gtc ttc acg ggc ggc gct ggc cgt gac tcgatc ctg ctg ggc gcc acg 2035 Val Phe Thr Gly Gly Ala Gly Arg Asp Ser IleLeu Leu Gly Ala Thr 630 635 640 645 acc aag gcg atc gtc atg ggc gcc ggcgac gac acc gtc acc gtc agc 2083 Thr Lys Ala Ile Val Met Gly Ala Gly AspAsp Thr Val Thr Val Ser 650 655 660 tcg gcg acc ctg ggc gct ggt ggt tcggtc aac ggc ggc gac ggc acc 2131 Ser Ala Thr Leu Gly Ala Gly Gly Ser ValAsn Gly Gly Asp Gly Thr 665 670 675 gac gtt ctg gtg gcc aac gtc aac ggttcg tcg ttc agc gct gac ccg 2179 Asp Val Leu Val Ala Asn Val Asn Gly SerSer Phe Ser Ala Asp Pro 680 685 690 gcc ttc ggc ggc ttc gaa acc ctc cgcgtc gct ggc gcg gcg gct caa 2227 Ala Phe Gly Gly Phe Glu Thr Leu Arg ValAla Gly Ala Ala Ala Gln 695 700 705 ggc tcg cac aac gcc aac ggc ttc acggct ctg caa ctg ggc gcg acg 2275 Gly Ser His Asn Ala Asn Gly Phe Thr AlaLeu Gln Leu Gly Ala Thr 710 715 720 725 gcg ggt gcg acg acc ttc acc aacgtt gcg gtg aat gtc ggc ctg acc 2323 Ala Gly Ala Thr Thr Phe Thr Asn ValAla Val Asn Val Gly Leu Thr 730 735 740 gtt ctg gcg gct ccg acc ggt acgacg acc gtg acc ctg gcc aac gcc 2371 Val Leu Ala Ala Pro Thr Gly Thr ThrThr Val Thr Leu Ala Asn Ala 745 750 755 acg ggc acc tcg gac gtg ttc aacctg acc ctg tcg tcc tcg gcc gct 2419 Thr Gly Thr Ser Asp Val Phe Asn LeuThr Leu Ser Ser Ser Ala Ala 760 765 770 ctg gcc gct ggt acg gtt gcg ctggct ggc gtc gag acg gtg aac atc 2467 Leu Ala Ala Gly Thr Val Ala Leu AlaGly Val Glu Thr Val Asn Ile 775 780 785 gcc gcc acc gac acc aac acg accgct cac gtc gac acg ctg acg ctg 2515 Ala Ala Thr Asp Thr Asn Thr Thr AlaHis Val Asp Thr Leu Thr Leu 790 795 800 805 caa gcc acc tcg gcc aag tcgatc gtg gtg acg ggc aac gcc ggt ctg 2563 Gln Ala Thr Ser Ala Lys Ser IleVal Val Thr Gly Asn Ala Gly Leu 810 815 820 aac ctg acc aac acc ggc aacacg gct gtc acc agc ttc gac gcc agc 2611 Asn Leu Thr Asn Thr Gly Asn ThrAla Val Thr Ser Phe Asp Ala Ser 825 830 835 gcc gtc acc ggc acg gct ccggct gtg acc ttc gtg tcg gcc aac acc 2659 Ala Val Thr Gly Thr Ala Pro AlaVal Thr Phe Val Ser Ala Asn Thr 840 845 850 acg gtg ggt gaa gtc gtc acgatc cgc ggc ggc gct ggc gcc gac tcg 2707 Thr Val Gly Glu Val Val Thr IleArg Gly Gly Ala Gly Ala Asp Ser 855 860 865 ctg acc ggt tcg gcc acc gccaat gac acc atc atc ggt ggc gct ggc 2755 Leu Thr Gly Ser Ala Thr Ala AsnAsp Thr Ile Ile Gly Gly Ala Gly 870 875 880 885 gct gac acc ctg gtc tacacc ggc ggt acg gac acc ttc acg ggt ggc 2803 Ala Asp Thr Leu Val Tyr ThrGly Gly Thr Asp Thr Phe Thr Gly Gly 890 895 900 acg ggc gcg gat atc ttcgat atc aac gct atc ggc acc tcg acc gct 2851 Thr Gly Ala Asp Ile Phe AspIle Asn Ala Ile Gly Thr Ser Thr Ala 905 910 915 ttc gtg acg atc acc gacgcc gct gtc ggc gac aag ctc gac ctc gtc 2899 Phe Val Thr Ile Thr Asp AlaAla Val Gly Asp Lys Leu Asp Leu Val 920 925 930 ggc atc tcg acg aac ggcgct atc gct gac ggc gcc ttc ggc gct gcg 2947 Gly Ile Ser Thr Asn Gly AlaIle Ala Asp Gly Ala Phe Gly Ala Ala 935 940 945 gtc acc ctg ggc gct gctgcg acc ctg gct cag tac ctg gac gct gct 2995 Val Thr Leu Gly Ala Ala AlaThr Leu Ala Gln Tyr Leu Asp Ala Ala 950 955 960 965 gct gcc ggc gac ggcagc ggc acc tcg gtt gcc aag tgg ttc cag ttc 3043 Ala Ala Gly Asp Gly SerGly Thr Ser Val Ala Lys Trp Phe Gln Phe 970 975 980 ggc ggc gac acc tatgtc gtc gtt gac agc tcg gct ggc gcg acc ttc 3091 Gly Gly Asp Thr Tyr ValVal Val Asp Ser Ser Ala Gly Ala Thr Phe 985 990 995 gtc agc ggc gct gacgcg gtg atc aag ctg acc ggt ctg gtc acg ctg 3139 Val Ser Gly Ala Asp AlaVal Ile Lys Leu Thr Gly Leu Val Thr Leu 1000 1005 1010 acc acc tcg gccttc gcc acc gaa gtc ctg acg ctc gcc taagcgaacg 3188 Thr Thr Ser Ala PheAla Thr Glu Val Leu Thr Leu Ala 1015 1020 1025 tctgatcctc gcctaggcgaggatcgctag actaagagac cccgtcttcc gaaagggagg 3248 cggggtcttt cttatgggcgctacgcgctg gccggccttg cctagttccg gt 3300 7 1026 PRT Caulobactercrescentus 7 Met Ala Tyr Thr Thr Ala Gln Leu Val Thr Ala Tyr Thr Asn AlaAsn 1 5 10 15 Leu Gly Lys Ala Pro Asp Ala Ala Thr Thr Leu Thr Leu AspAla Tyr 20 25 30 Ala Thr Gln Thr Gln Thr Gly Gly Leu Ser Asp Ala Ala AlaLeu Thr 35 40 45 Asn Thr Leu Lys Leu Val Asn Ser Thr Thr Ala Val Ala IleGln Thr 50 55 60 Tyr Gln Phe Phe Thr Gly Val Ala Pro Ser Ala Ala Gly LeuAsp Phe 65 70 75 80 Leu Val Asp Ser Thr Thr Asn Thr Asn Asp Leu Asn AspAla Tyr Tyr 85 90 95 Ser Lys Phe Ala Gln Glu Asn Arg Phe Ile Asn Phe SerIle Asn Leu 100 105 110 Ala Thr Gly Ala Gly Ala Gly Ala Thr Ala Phe AlaAla Ala Tyr Thr 115 120 125 Gly Val Ser Tyr Ala Gln Thr Val Ala Thr AlaTyr Asp Lys Ile Ile 130 135 140 Gly Asn Ala Val Ala Thr Ala Ala Gly ValAsp Val Ala Ala Ala Val 145 150 155 160 Ala Phe Leu Ser Arg Gln Ala AsnIle Asp Tyr Leu Thr Ala Phe Val 165 170 175 Arg Ala Asn Thr Pro Phe ThrAla Ala Ala Asp Ile Asp Leu Ala Val 180 185 190 Lys Ala Ala Leu Ile GlyThr Ile Leu Asn Ala Ala Thr Val Ser Gly 195 200 205 Ile Gly Gly Tyr AlaThr Ala Thr Ala Ala Met Ile Asn Asp Leu Ser 210 215 220 Asp Gly Ala LeuSer Thr Asp Asn Ala Ala Gly Val Asn Leu Phe Thr 225 230 235 240 Ala TyrPro Ser Ser Gly Val Ser Gly Ser Thr Leu Ser Leu Thr Thr 245 250 255 GlyThr Asp Thr Leu Thr Gly Thr Ala Asn Asn Asp Thr Phe Val Ala 260 265 270Gly Glu Val Ala Gly Ala Ala Thr Leu Thr Val Gly Asp Thr Leu Ser 275 280285 Gly Gly Ala Gly Thr Asp Val Leu Asn Trp Val Gln Ala Ala Ala Val 290295 300 Thr Ala Leu Pro Thr Gly Val Thr Ile Ser Gly Ile Glu Thr Met Asn305 310 315 320 Val Thr Ser Gly Ala Ala Ile Thr Leu Asn Thr Ser Ser GlyVal Thr 325 330 335 Gly Leu Thr Ala Leu Asn Thr Asn Thr Ser Gly Ala AlaGln Thr Val 340 345 350 Thr Ala Gly Ala Gly Gln Asn Leu Thr Ala Thr ThrAla Ala Gln Ala 355 360 365 Ala Asn Asn Val Ala Val Asp Gly Arg Ala AsnVal Thr Val Ala Ser 370 375 380 Thr Gly Val Thr Ser Gly Thr Thr Thr ValGly Ala Asn Ser Ala Ala 385 390 395 400 Ser Gly Thr Val Ser Val Ser ValAla Asn Ser Ser Thr Thr Thr Thr 405 410 415 Gly Ala Ile Ala Val Thr GlyGly Thr Ala Val Thr Val Ala Gln Thr 420 425 430 Ala Gly Asn Ala Val AsnThr Thr Leu Thr Gln Ala Asp Val Thr Val 435 440 445 Thr Gly Asn Ser SerThr Thr Ala Val Thr Val Thr Gln Thr Ala Ala 450 455 460 Ala Thr Ala GlyAla Thr Val Ala Gly Arg Val Asn Gly Ala Val Thr 465 470 475 480 Ile ThrAsp Ser Ala Ala Ala Ser Ala Thr Thr Ala Gly Lys Ile Ala 485 490 495 ThrVal Thr Leu Gly Ser Phe Gly Ala Ala Thr Ile Asp Ser Ser Ala 500 505 510Leu Thr Thr Val Asn Leu Ser Gly Thr Gly Thr Ser Leu Gly Ile Gly 515 520525 Arg Gly Ala Leu Thr Ala Thr Pro Thr Ala Asn Thr Leu Thr Leu Asn 530535 540 Val Asn Gly Leu Thr Thr Thr Gly Ala Ile Thr Asp Ser Glu Ala Ala545 550 555 560 Ala Asp Asp Gly Phe Thr Thr Ile Asn Ile Ala Gly Ser ThrAla Ser 565 570 575 Ser Thr Ile Ala Ser Leu Val Ala Ala Asp Ala Thr ThrLeu Asn Ile 580 585 590 Ser Gly Asp Ala Arg Val Thr Ile Thr Ser His ThrAla Ala Ala Leu 595 600 605 Thr Gly Ile Thr Val Thr Asn Ser Val Gly AlaThr Leu Gly Ala Glu 610 615 620 Leu Ala Thr Gly Leu Val Phe Thr Gly GlyAla Gly Arg Asp Ser Ile 625 630 635 640 Leu Leu Gly Ala Thr Thr Lys AlaIle Val Met Gly Ala Gly Asp Asp 645 650 655 Thr Val Thr Val Ser Ser AlaThr Leu Gly Ala Gly Gly Ser Val Asn 660 665 670 Gly Gly Asp Gly Thr AspVal Leu Val Ala Asn Val Asn Gly Ser Ser 675 680 685 Phe Ser Ala Asp ProAla Phe Gly Gly Phe Glu Thr Leu Arg Val Ala 690 695 700 Gly Ala Ala AlaGln Gly Ser His Asn Ala Asn Gly Phe Thr Ala Leu 705 710 715 720 Gln LeuGly Ala Thr Ala Gly Ala Thr Thr Phe Thr Asn Val Ala Val 725 730 735 AsnVal Gly Leu Thr Val Leu Ala Ala Pro Thr Gly Thr Thr Thr Val 740 745 750Thr Leu Ala Asn Ala Thr Gly Thr Ser Asp Val Phe Asn Leu Thr Leu 755 760765 Ser Ser Ser Ala Ala Leu Ala Ala Gly Thr Val Ala Leu Ala Gly Val 770775 780 Glu Thr Val Asn Ile Ala Ala Thr Asp Thr Asn Thr Thr Ala His Val785 790 795 800 Asp Thr Leu Thr Leu Gln Ala Thr Ser Ala Lys Ser Ile ValVal Thr 805 810 815 Gly Asn Ala Gly Leu Asn Leu Thr Asn Thr Gly Asn ThrAla Val Thr 820 825 830 Ser Phe Asp Ala Ser Ala Val Thr Gly Thr Ala ProAla Val Thr Phe 835 840 845 Val Ser Ala Asn Thr Thr Val Gly Glu Val ValThr Ile Arg Gly Gly 850 855 860 Ala Gly Ala Asp Ser Leu Thr Gly Ser AlaThr Ala Asn Asp Thr Ile 865 870 875 880 Ile Gly Gly Ala Gly Ala Asp ThrLeu Val Tyr Thr Gly Gly Thr Asp 885 890 895 Thr Phe Thr Gly Gly Thr GlyAla Asp Ile Phe Asp Ile Asn Ala Ile 900 905 910 Gly Thr Ser Thr Ala PheVal Thr Ile Thr Asp Ala Ala Val Gly Asp 915 920 925 Lys Leu Asp Leu ValGly Ile Ser Thr Asn Gly Ala Ile Ala Asp Gly 930 935 940 Ala Phe Gly AlaAla Val Thr Leu Gly Ala Ala Ala Thr Leu Ala Gln 945 950 955 960 Tyr LeuAsp Ala Ala Ala Ala Gly Asp Gly Ser Gly Thr Ser Val Ala 965 970 975 LysTrp Phe Gln Phe Gly Gly Asp Thr Tyr Val Val Val Asp Ser Ser 980 985 990Ala Gly Ala Thr Phe Val Ser Gly Ala Asp Ala Val Ile Lys Leu Thr 995 10001005 Gly Leu Val Thr Leu Thr Thr Ser Ala Phe Ala Thr Glu Val Leu Thr1010 1015 1020 Leu Ala 1025 8 17 PRT Pseudomonas aeroginosa VARIANT(1)...(17) Xaa = Any Amino Acid 8 Xaa Cys Thr Ser Asp Gln Asp Glu GlnPhe Ile Pro Lys Gly Cys Ser 1 5 10 15 Xaa 9 184 PRT InfectiousHematopoietic Nervosis Virus 9 Glu Tyr Asn Ser Gly Ala Glu Ile Leu SerPhe Pro Lys Cys Glu Asp 1 5 10 15 Lys Thr Met Gly Met Arg Gly Asn LeuAsp Asp Phe Ala Tyr Leu Asp 20 25 30 Asp Leu Val Lys Ala Ser Glu Ser ArgGlu Glu Cys Leu Glu Ala His 35 40 45 Ala Glu Ile Ile Ser Thr Asn Ser ValThr Pro Tyr Leu Leu Ser Lys 50 55 60 Phe Arg Ser Pro His Pro Gly Ile AsnAsp Val Tyr Ala Met His Lys 65 70 75 80 Gly Ser Ile Tyr His Gly Met SerMet Thr Val Ala Val Asp Glu Val 85 90 95 Ser Lys Asp Arg Thr Thr Tyr ArgAla His Arg Ala Thr Ser Phe Thr 100 105 110 Lys Trp Glu Arg Pro Phe GlyAsp Glu Trp Glu Gly Phe His Gly Leu 115 120 125 His Gly Asn Asn Thr ThrIle Ile Pro Asp Leu Glu Lys Tyr Val Ala 130 135 140 Gln Tyr Lys Thr SerMet Met Glu Pro Met Ser Ile Lys Ser Val Pro 145 150 155 160 His Pro SerIle Leu Ala Phe Tyr Asn Glu Thr Asp Leu Ser Gly Ile 165 170 175 Ser IleArg Lys Leu Asp Ser Phe 180 10 12 DNA Artificial Sequence Syntheticallygenerated linker 10 cgacggatcc gt 12 11 17 PRT Artificial SequenceSynthetically generated peptide 11 Ala Glu Ala Ala Ala Lys Glu Ala AlaAla Lys Cys Ala Ala Ala His 1 5 10 15 Ala 12 552 DNA InfectiousHematopoietic Nervosis Virus CDS (1)...(552) 12 gaa tac aat tct gga gcagaa atc ctc tcg ttc ccg aag tgt gag gac 48 Glu Tyr Asn Ser Gly Ala GluIle Leu Ser Phe Pro Lys Cys Glu Asp 1 5 10 15 aag acg atg ggg atg agggga aac ttg gat gac ttt gcc tat cta gac 96 Lys Thr Met Gly Met Arg GlyAsn Leu Asp Asp Phe Ala Tyr Leu Asp 20 25 30 gat ctg gtg aag gcc tct gagagc aga gag gaa tgt ctt gag gcg cac 144 Asp Leu Val Lys Ala Ser Glu SerArg Glu Glu Cys Leu Glu Ala His 35 40 45 gcc gag ata ata tca aca aac agtgtg act cca tac ctc cta tcc aag 192 Ala Glu Ile Ile Ser Thr Asn Ser ValThr Pro Tyr Leu Leu Ser Lys 50 55 60 ttc cga tct cca cat ccc gga ata aatgac gtc tac gct atg cac aaa 240 Phe Arg Ser Pro His Pro Gly Ile Asn AspVal Tyr Ala Met His Lys 65 70 75 80 ggc tcc atc tat cac ggg atg tcc atgacg gtc gct gtg gac gag gta 288 Gly Ser Ile Tyr His Gly Met Ser Met ThrVal Ala Val Asp Glu Val 85 90 95 tcc aag gac agg acg acg tac agg gcc catcgc gct acc agc ttc acg 336 Ser Lys Asp Arg Thr Thr Tyr Arg Ala His ArgAla Thr Ser Phe Thr 100 105 110 aaa tgg gaa cga ccc ttt ggg gat gag tgggag ggc ttt cac gga ttg 384 Lys Trp Glu Arg Pro Phe Gly Asp Glu Trp GluGly Phe His Gly Leu 115 120 125 cac gga aac aac acc acc att att cca gacctg gag aaa tac gtc gcc 432 His Gly Asn Asn Thr Thr Ile Ile Pro Asp LeuGlu Lys Tyr Val Ala 130 135 140 cag tac aag acg agc atg atg gaa ccg atgagc atc aaa tcc gta ccc 480 Gln Tyr Lys Thr Ser Met Met Glu Pro Met SerIle Lys Ser Val Pro 145 150 155 160 cat cca agc atc ctg gcc ttc tac aatgag aca gac tta tca ggg atc 528 His Pro Ser Ile Leu Ala Phe Tyr Asn GluThr Asp Leu Ser Gly Ile 165 170 175 tcc atc agg aaa ttg gac tca ttc 552Ser Ile Arg Lys Leu Asp Ser Phe 180

We claim:
 1. A nucleic acid comprising: a nucleotide sequence encoding apolypeptide consisting of a part of a Caulobacter crescentus S-layerprotein, the part comprising at least the 82 C-terminal amino acids ofthe S-layer protein and does not contain amino acids 1-29 of the S-layerprotein; and one or more restriction sites adjacent to or within thenucleotide sequence.
 2. A nucleic acid comprising a sequence encoding afusion polypeptide, the fusion polypeptide consisting of: a part of aCaulobacter crescentus S-layer protein, the part comprising at least the82 C-terminal amino acids of the S-layer protein and does not containamino acids 1-29 of the S-layer protein; and an amino acid sequenceheterologous to the S-layer protein and adjacent to or within the part.3. The nucleic acid of claim 1, wherein the part comprises amino acidscorresponding to about amino acids 945-1026 of SEQ ID NO:7.
 4. Thenucleic acid of claim 2, wherein the part comprises amino acidscorresponding to about amino acids 945-1026 of SEQ ID NO:7.
 5. Thenucleic acid of claim 1, wherein the part comprises amino acidscorresponding to about amino acids 850-1026 of SEQ ID NO:7.
 6. Thenucleic acid of claim 2, wherein the part comprises amino acidscorresponding to about amino acids 850-1026 of SEQ ID NO:7.
 7. Thenucleic acid of claim 1, wherein the part comprises amino acidscorresponding to about amino acids 782-1026 of SEQ ID NO:7.
 8. Thenucleic acid of claim 2, wherein the part comprises amino acidscorresponding to about amino acids 782-1026 of SEQ ID NO:7.
 9. Thenucleic acid of claim 2, wherein the amino acid sequence heterologous tothe S-layer protein comprises one or more polypeptides of up to 200amino acids in length.
 10. A bacterial cell comprising the nucleic acidof claim
 2. 11. The cell of claim 10, wherein the cell is a member ofthe genus Caulobacter.
 12. The cell of claim 11, wherein the nucleicacid further comprises a promoter operably linked to the sequenceencoding the fusion polypeptide, and the fusion polypeptide is expressedin the cell and secreted from the cell.
 13. The cell of claim 12,wherein the cell forms a S-layer comprising the fusion polypeptide on asurface of the cell.
 14. The cell of claim 10, wherein the amino acidsequence heterologous to the S-layer protein comprises one or morepolypeptides of up to 60 amino acids in length.
 15. A bacterial cellcomprising the nucleic acid of claim
 4. 16. A bacterial cell comprisingthe nucleic acid of claim
 6. 17. A bacterial cell comprising the nucleicacid of claim
 8. 18. A bacterial cell comprising the nucleic acid ofclaim
 9. 19. A fusion polypeptide obtained from a cell surface or cellmedium of a culture comprising the cell of claim 12, wherein the fusionpolypeptide consists of: a part of a Caulobacter crescentus S-layerprotein, the part comprising at least the 82 C-terminal amino acids ofthe S-layer protein and does not contain amino acids 1-29 of the S-averprotein; and an amino acid sequence heterologous to the S-layer proteinand adjacent to or within the part, wherein the amino acid sequencecomprises one or more polypeptides of up to about 200 amino acids inlength.
 20. The fusion polypeptide of claim 19, wherein the amino acidsequence comprises one or more polypeptides of up to about 60 aminoacids in length.
 21. The fusion polypeptide of claim 19, wherein thepart comprises amino acids corresponding to about amino acids 945-1026of SEQ ID NO:7.
 22. The fusion polypeptide of claim 21, wherein the partcomprises amino acids corresponding to about amino acids 782-944 of SEQID NO:7.
 23. The fusion polypeptide of claim 19, wherein the amino acidsequence comprises one or more copies of all or part of SEQ ID NO:9.